Enzymes immobilized on solid-phase matrices have found various applications in biotechnology, molecular biology and molecular diagnostics and can serve as industrial catalysts and as specific reagents for analytical procedures. A wide range of supports have been utilized for immobilization among which particle-based supports are the most commonly implemented. Type of support used for immobilization is one of the key considerations in practical application due to different immobilization efficiency, ligand utilization and the mass transfer regime. The mass transfer between the mobile and the particulate stationary phase is often a bottleneck for the entire process due to slow pore diffusion of large molecules. In contrast, monoliths due to their structure enable almost flow-independent properties. Consequently, the overall behavior of the immobilized ligand reflects its intrinsic reaction kinetics. Therefore, such an immobilized system is expected to allow higher throughput because of higher enzyme efficiency, especially pronounced for macromolecular substrates having low mobility. In this work, different methods for immobilization of enzymes on Convective Interaction Media monolithic supports are presented. In particular, enzymes acting on macromolecular substrates, such as trypsin, deoxyribonuclease and ribonuclease, are described in detail. Immobilized efficiency is evaluated for different immobilization procedures in terms of biologic activity and long-term stability. Finally, their performance on real samples is demonstrated.